Exploration into the molecular forces shaping the evolution of Tetrodotoxin resistance in garter snakes

A main objective of evolutionary biology is the exploration of the mechanisms of adaptation from a molecular perspective. Many adaptive traits constitute complex phenotypes involving several gene products. Toxin resistance can be considered a complex phenotype when multiple proteins are each targeted by a specific toxin. Tetrodotoxin (TTX) resistance in garter snakes (Thamnophis sirtalis) adapted to consuming toxic prey fits this model of a complex phenotype because the potent neurotoxin TTX targets multiple proteins within snake tissues. These TTX target proteins in resistant T. sirtalis are paralogous voltage-gated sodium channels (NaV), which evolved amino acid substitutions that decrease binding affinity of TTX. To better understand evolution of TTX resistance in NaV channels, I reviewed examples of resistant channels across a wide range of taxonomic groups encompassing vertebrate and invertebrate species. This exploration revealed a high degree of molecular convergence in the protein changes that confer resistance. This convergence likely results from a combination of the binding specificity of TTX and functional constraints in NaV that are shared among unrelated taxa.
Evolution of physiological TTX resistance in T. sirtalis seems to require the acquisition of TTX-resistant substitutions in multiple NaV paralogs. The resistance-related changes found in one paralog expressed in peripheral nerves (NaV1.7) show ancient evolution. The substitutions in NaV1.7 were acquired before garter snakes evolved to consume TTX-bearing prey. In fact, these substitutions are found in most snakes, suggesting that these changes were acquired to serve a function unrelated to TTX resistance. I used channel expression in a heterologous system and electrophysiology to explore the hypothesis that evolution of ancient resistance in NaV1.7 is a by-product of ancestral selection for biophysical changes in this paralog. Evolution of resistance in the skeletal muscle paralog (NaV1.4) of T. sirtalis shows signs of recent evolution in direct response to TTX exposure. Different snake populations exposed to TTX-bearing prey present few variants of NaV1.4 exhibiting TTX-resistant amino acid substitutions. However, there is a dearth of intermediate channel variants between the ancestral TTX-sensitive NaV1.4 (lacking substitutions) and the derived channel forms found in highly resistant snakes. This implies that selection may disfavor intermediate forms of the channel, possibly due to molecular constraints limiting disadvantageous combinations of channel substitutions. I investigated molecular constraint in the evolution of TTX resistance in NaV1.4 by recreating the intermediate steps in one possible mutational trajectory from a sensitive channel to a TTX-resistant channel variant. I used electrophysiology to assess TTX resistance and different aspects of channel function. This work helped reveal pleiotropic effects of amino acid mutations involved in the evolution of TTX resistance in NaV1.4. The exploration of molecular evolution in two of the paralogs contributing to physiological resistance in T. sirtalis can help us understand how molecular factors shaping evolution of proteins influence evolution of a complex trait.